Process for facilitating nucleic acid transfer

ABSTRACT

Means for transferring efficiently a desired nucleic acid into a cell is provided. 
     The present invention comprises using a complex comprising a collagen or a collagen derivative, and a desired nucleic acid.

RELATED APPLICATIONS

This application is a Divisional of co-pending Application No.10/481,511 filed on Dec. 19, 2003, and for which priority is claimedunder 35 U.S.C. §120; and this application claims priority ofApplication No. 2001-186320 filed in Japan on Jun. 20, 2001 and forwhich priority is claimed under 35 U.S.C. §119; and this applicationclaims priority of Application No. 2001-278293 filed in Japan on Sep.13, 2001, and for which priority is claimed under 35 U.S.C. §119; andthis application claims priority of Application No. PCT/JP02/06137 filedin Japan on Jun. 20, 2002, and for which priority is claimed under 35U.S.C. §119, for which the entire contents of all are herebyincorporated by referenced.

FIELD OF THE INVENTION

The present invention belongs to medical field, specifically to genetherapy and genetic fundamental research. More specifically, theinvention relates to preparations for facilitating the transfer of adesired nucleic acid into a target cell, and processes therefor.

BACKGROUND ART

Recently, gene therapy has been actively studied, and appliedpractically to clinical therapy of various cancers and genetic diseases.Gene therapy is an approach to treat a disease by repairing orcorrecting a defective gene, and comprises transferring a gene encodingan intended enzyme, cytokine, or the like into a cell of a patient, andallowing to produce the intended substance from the gene in the body,thereby treating the disease. Gene therapy is a medication that controlsa basis of life, and has a potential to treat various diseases such asAIDS, rheumatoid arthritis, lifestyle-related diseases, in addition tocancers and genetic diseases.

In gene therapy, transfer efficiency of gene into a target cell is animportant factor in the increased efficacy of the therapy. Gene therapyfor cancers includes therapies by virus such as adenovirus(Cardiovascular Research, 28, 445 (1994); Science, 256, 808 (1992);Gastroenterology, 106, 1076 (1994); TIBTECH, 11, 182 (1993); J. Biol.Chem, 266, 3361 (1991); Nature Medicine, 1, 583 (1995) and the citedreferences therein) and those by liposome formulations (Biochem BiophysActa, 1097, 1 (1991); Human Gene Therapy, 3, 399 (1992); Proc. Natl.Acad. Sci. USA, 89, 11277 (1992)). Transfer efficiency of genes isgenerally higher in therapy using virus vectors than therapy usingliposome formulations. However, therapy using virus vectors suffers froma problem that multiple administrations are hardly conducted due toimmunological responses to viruses (J. Biol. Chem., 269, 13695(1994),Am. J. Respir. Cell Mol. Biol., 10, 369 (1994)).

On the other hand, since the analysis on the whole human geneticinformation (human genome) was almost completed, the focus has beenshifted to post-genome strategies how to utilize the accumulated humangenetic information in the fields of medication and industry.Specifically, examinations on human gene functions, as well as thestructures and functions of the proteins encoded by the gene using theanalyzed genetic information have been emphasized. Such a post-genomeexaminations require the expression and the production of proteins,which necessarily involve the transfer of intended genes into cells.Genes to be transferred into host cells by adenovirus vectors andliposome vectors or plasmid DNA vectors are not integrated into thegenome of the cells, and are transiently expressed. Such vectors can notaccomplish the constitutive expression of the genes, which is importantin gene therapy and analysis on gene functions.

DISCLOSURE OF THE INVENTION

Thus, an approach to efficiently transfer a nucleic acid representing agene into a desired cell, and to express the gene during a long periodof time without integration of the gene into chromosome of host cells isexpected to provide a great utility.

The inventors of the present application found that collagens have anunexpected action, and created an approach to efficiently transfer anucleic acid into a desired cell. Specifically, we found that thecontact of a collagen and a nucleic acid such as plasmid DNAsurprisingly results in the formation of a complex, and the formation ofa complex facilitates the transfer of a nucleic acid into a cell andexpresses the gene during a long period of time. Although JapanesePatent Publication (kokai) No. 71542/1997 describes formulationscontaining a gene wherein the gene is comprised in a carrier of abiocompatible material such as a collagen, the formulations aresustained release formulations that gradually releases the gene in aliving body.

The invention is based on the newly founded use of a collagen or acollagen derivative.

More specifically, the invention relates to:

(1) A preparation for facilitating the transfer of a nucleic acid into atarget cell, which comprises a collagen or a collagen derivative;

(2) A preparation for facilitating the transfer of a nucleic acid into atarget cell, which comprises a collagen or a collagen derivativecomplexed with a desired nucleic acid, preferably a preparation forfacilitating the transfer of a nucleic acid, wherein the complex is in aform of particle, more preferably a preparation for facilitating thetransfer of a nucleic acid, wherein the major axis of the particle is300 nm to 300 μm, preferably 300 nm to 100 μm, more preferably 300 nm to50 μm, even more preferably 300 nm to 30 μm; Specifically, a preparationfor facilitating the transfer of a nucleic acid wherein the desirednucleic acid is a plasmid DNA, and wherein the ratio of the number of acollagen molecule or a collagen derivative molecule to the number of anucleotide monomer of the plasmid DNA in the complex is 1:20 to 1: thenumber of a nucleotide monomer of the plasmid DNA, preferably 1:50 to 1:the number of a nucleotide monomer of the plasmid DNA, more preferably1:50 to 1:4000, still more preferably 1:50 to 1:2000, and still morepreferably 1:50 to 1:1000, or a preparation for facilitating thetransfer of a nucleic acid wherein the nucleic acid is anoligonucleotide, and which the ratio of the number of a collagenmolecule or a collagen derivative molecule to the number of a nucleotidemonomer of the oligonucleotide in the complex is 1:1 to 1:200,preferably 1:3 to 1:150, more preferably 1:20 to 1:120, and still morepreferably 1:50 to 1:120;

(3) A particle of the complex comprising a collagen or a collagenderivative and a desired nucleic acid;

(4) A process for preparing a particle of the complex according to thepresent invention, which comprises mixing a collagen or a collagenderivative and a desired nucleic acid in a solution comprising an agentthat inhibits the formation of collagen association body;

(5) A medical instrument, of which the surface is coated with a particleof the complex according to above (3) or a cell culture instrument, ofwhich the surface is coated with the particle of the complex;

(6) A process for transferring a desired nucleic acid into a target cellor a process for improving the expression level of a desired nucleicacid in a target cell, which comprises using a particle of the complexaccording to above (3);

(7) A process for examining the function of a gene or a protein in atarget cell, which comprises coating a solid surface with a particle ofthe complex according to above (3) that comprises the gene, a geneencoding the protein, or a nucleic acid inhibiting the expression of thegene or the protein in a cell; culturing the target cell on the solidsurface; and examining the expression level of the nucleic acid or theexpression level of the gene or the protein in the target cell, or theproliferation ratio or the phenotype of the cell; and

(8) A process for screening for a nucleic acid that treats a disease,which comprises coating a solid surface with a particle of the complexaccording to above (3) that comprises a nucleic acid candidate thatinhibits the expression of a gene associated with the disease in a cell;culturing the cell presenting the condition of the disease on the solidsurface; and examining the expression level of the gene to be inhibitedwith each of the nucleic acid candidate, or the proliferation ratio orthe phenotype of the cell.

The working examples hereinafter illustrate that the preparations forfacilitating the transfer of a nucleic acid according to the presentinvention improved the transfer efficiency of gene into a target cell asshown to express the nucleic acid in a cell culture system in vitrowhere the gene expression is not observed by mere plasmid DNA. Further,those examples illustrate that the preparations for facilitating thetransfer of a nucleic acid according to the present invention increasedthe stability of a nucleic acid within a cell as shown to sustain theexpression of the nucleic acid during a longer period of time thanliposome formulations.

According to the invention, it has been found that a collagen isinteracted electrostatically and/or physically with a nucleic acid toform a complex. Thus, it is believed that the sustained expression of anucleic acid as observed in the working examples would result from thecomplex formation leading to the increased stability of nucleic acidswithin cells. This is quite different from the mechanism of thesustained release of gene by collagens that was conventionallyunderstood that a gene encapsulated in collagen matrix is graduallyreleased according to the biological degradation of collagen.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a photograph substitute for drawing which depicts anagarose-gel electrophoresis showing the electrostatic interactionbetween the defined concentration of plasmid DNA and the variousconcentrations of atelocollagen.

FIG. 2 is a photograph substitute for drawing which depicts anagarose-gel electrophoresis showing the effect of sodium chloride on theelectrostatic interaction between plasmid DNA and atelocollagen.

FIG. 3 is a photograph substitute for drawing which depicts anagarose-gel electrophoresis showing the effect of heparan sulfate on theelectrostatic interaction between plasmid DNA and atelocollagen.

FIG. 4 is a micrograph showing a form of the complexes between plasmidDNA and atelocollagen in various concentrations.

FIG. 5 is a micrograph showing a form of the complexes between plasmidDNA and atelocollagen in various concentrations that were stored for aweek.

FIG. 6 is a graph showing a comparison in the duration time of geneexpression among the atelocollagen gel formulation, the cationicliposome formulation, and the plasmid DNA in a PBS solution.

FIG. 7 is a graph showing the relationship between the complexescomprising a collagen in various concentrations and the transferefficiency of plasmid DNA seven days after the transfection by dropwiseaddition.

FIG. 8 is a graph showing a fluorescence intensity representing thetransfer efficiency of plasmid DNA in the complexes comprising acollagen in various concentrations seven days after the transfection.

FIG. 9 is a graph showing the relationship between the complexescomprising a collagen in various concentrations and the transferefficiency of plasmid DNA seven days after the transfection by solidcoating.

FIG. 10 is a graph showing the relationship between the complexescomprising a plasmid DNA in various concentrations and the transferefficiency of plasmid DNA seven days after the transfection by dropwiseaddition.

FIG. 11 is a graph showing the relationship between the complexescomprising a plasmid DNA in various concentrations and the transferefficiency of plasmid DNA seven days after the transfection by solidcoating.

FIG. 12 is a graph showing a fluorescence intensity representing thetransfer efficiency of plasmid DNA seven days after the transfection bydropwise addition, and a micrograph showing the fluorescence.

FIG. 13 is a graph showing inhibitory effects of the present inventionon the cell proliferation.

FIG. 14 is a graph showing that adenovirus was transferred by solidcoating in a dose-dependent manner, and a micrograph showing thefluorescence.

FIG. 15 is a graph showing the relationship between the number ofcollagen bound to one molecule of plasmid DNA and the average major axisof the complexes.

FIG. 16 is a graph showing the relationship between the number ofnucleotide monomer of desired nucleic acids per collagen molecule andthe average major axis of the complexes.

FIG. 17 is a graph showing the relationship between the molecular numberratio of oligonucleotide to collagen at the time of the mixture and thenumber of oligonucleotide bound to one molecule of collagen in thecomplex.

FIG. 18 is a fluorescence micrograph obtained by observing the complexescomprising the oligonucleotides released from the surface of the cellculture instrument according to the present invention with fluorescencemicroscopy.

FIG. 19 is a fluorescence micrograph obtained by observing the complexescomprising the plasmid DNA released from the surface of the cell cultureinstrument according to the present invention with fluorescencemicroscopy.

BEST MODE FOR CARRYING OUT THE INVENTION 1) A Preparation forFacilitating the Transfer of a Nucleic Acid

As the first embodiment, the invention provides a preparation forfacilitating the transfer of a nucleic acid into a target cell, whichcomprises a collagen or a collagen derivative. The embodiment is basedon the effect of a collagen or a collagen derivative on the facilitationof the transfer of a nucleic acid into a target cell, which has beenfound for the first time. In other words, the present embodiment of theinvention provides a new use of a collagen or a collagen derivative tofacilitate the transfer of a nucleic acid into a target cell.

As used herein, “a collagen or a collagen derivative” generally meansany kind of collages or collagen derivatives as used in medical,cosmetic, industrial, and food fields. A soluble collagen or asolubilized collagen is preferably utilized. Soluble collagens aresoluble in an acidic or neutral water or a water containing a salt,whereas solubilized collagens include an enzymatically solubilizedcollagen which may be solubilized with an enzyme, an alkali-solubilizedcollagen which may be solubilized with an alkali, both collagens beingpreferably capable of penetrating through a membrane filter having apore size of 1 micrometer. Solubility of collagen varies depending onthe crosslinking degree of the collagen, and higher is the crosslinkingdegree, more difficult the collagen is solubilized. Accordingly, thecrosslinking degree of a collagen as used in the present invention is,for example, not more than trimer, more preferably not more than dimer.Preferable molecular weight of the collagen is, for example, from about300,000 to about 900,000, and more preferably from about 300,000 toabout 600,000. Collagens as used herein include those extracted from anyanimal species, and it is desired that preferable collagens areextracted from vertebrates, more preferable collagens are extracted froma mammal, a bird, or a fish, and still more preferable collagens areextracted from a mammal or a bird having a high denaturationtemperature. Any type of collagen may be used, and, because of the typeexisting in animal bodies, type I-V collagens are preferable. Forexample, such collagens include a type I collagen obtained by acidextraction from a mammal dermis, and, more preferably, they include, forexample, a type I collagen obtained by acid extraction from calf dermis,a type I collagen produced by genetic engineering, and the like.Collagens derived from tendon, which are also type I collagens, are notsuitable because they have a high degree of crosslinking and areinsoluble. Further, an atelocollagen that is obtained by removingenzymatically a telopeptide having a high antigenicity or anatelocollagen produced by genetic engineering is preferable for the sakeof safety, and an atelocollagen having three or less tyrosine residuesper 1000 residues is more preferable. Alternatively, collagens having amodified side chain, crosslinked collagens or the like may be utilizedif desired. Collagens having a modified side chain includes, forexample, succinylated collagens and methylated collagens, whereascrosslinked collagens include, for example, collagens treated withglutaraldehyde, hexamethylene diisocyanat, a polyepoxy compound or thelike (Fragrance Journal 1989-12, 104-109, Japanese PatentPublication(kokai) No. 59522/1995). Preferred collagen derivatives are agelatin or a gelatin-crosslinking complex, or a crosslinking complexthereof with a collagen.

Collagens or collagen derivatives may be used in admixture with anotherbiocompatible material. Biocompatible materials include, for example,gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate,chitin, chitosan, alginic acid, pectin, agarose, hydroxyapatite,polypropylenes, polyethylenes, polydimethylsiloxane, and a polymer ofglycolic acid, lactic acid or amino acid, and a copolymer thereof, and amixture containing two or more of those biocompatible materials.

2) A Preparation for Facilitating the Transfer of a Nucleic Acid, WhichComprises a Complex

As the second embodiment, the present invention provides a preparationfor facilitating the transfer of a nucleic acid into a target cell,which comprises a collagen or a collagen derivative and a desirednucleic acid, preferably comprises particles of a complex as anessential component.

Nucleic acids are hardly transferred into cells when administered to thecells in vitro solely in the presence of blood serum. Nucleic acids canbe efficiently transferred into cells when formed with a collagen or acollagen derivative into a complex.

As used herein, “a nucleic acid” may be any polynucleotide or anyoligonucleotide, and may be any DNA or RNA molecule. DNA moleculesinclude a plasmid DNA, cDNA, a genomic DNA or a synthesized DNA. BothDNA and RNA may be double-stranded or single-stranded. Single-strandedones include a coding strand and a non-cording strand. As used herein,“a nucleic acid” includes a DNA derivative and an RNA derivative, whichderivative means a nucleic acid having a phosphorothioate bond, or anucleic acid containing an internucleotide having a phosphate, sugar orbase moiety chemically modified to avoid enzymatic degradations. As usedherein, “a nucleic acid” also includes viruses such as adenovirus andretrovirus.

Preferably, “a nucleic acid” is an oligonucleotide or a ribozyme, andmore preferably an oligonucleotide or a ribozyme that is from 5 to 100mer, more preferably from 5 to 30 mer. It is preferred to utilize aplasmid DNA encoding a protein exhibiting a physiological activity totreat or ameliorate pathological conditions or a plasmid DNA encoding aprotein inducing an immunological response to treat or amelioratepathological conditions.

When the nucleic acid is a vector as used in gene therapy such as aplasmid DNA or a virus, it is preferably a system as constructed toexpress the encoded genetic information in cells, such as a vector thatcomprises an element such as a promoter necessary to express an intendedgene, or an element capable of integrating into chromosomes. Size ofplasmid DNAs as a nucleic acid used herein is not limited, and may beselected appropriately from the sizes that allow the encoded geneticinformation to be efficiently prepared via genetic engineering, andefficiently expressed in cells to be transferred.

The preparation for facilitating the transfer of a nucleic acidaccording to the invention may contain a few kinds of separate vectorsincorporated with different desired nucleic acids. Further, a vector maycomprise many genetic information. Amounts of the vector comprised inthe preparation for facilitating the transfer are not limited.

Nucleic acids encoding a protein necessary to be expressed in genetherapy includes any gene capable to be used in the treatment of agenetic disease, which is exemplified by, but is not limited to, a geneencoding an enzyme such as adenosine deaminase, thymidine kinase; acytokine such as GM-CSF, IL-2; or fibroblast growth factor HST-1 (FGF4).Nucleic acids encoding other proteins necessary to be expressed in genetherapy includes, but is not limited to, a gene aimed at the treatmentor the prevention for an infection or a tumor, which encodes a proteinor a peptide serving as an antigen to induce immune response, i.e., thegene encoding the protein or the peptide capable of serving as anantigen such as mentioned above, for example, a gene encoding thesurface protein HA or NA, or the nuclear protein NP of influenza virus,type C hepatitis virus E2 or NS 1 protein, type B hepatitis virus HBsantigen protein, type A hepatitis virus capsid protein VP1 or VP3 orcapsidoid protein, dengue virus Egp protein, RS virus F or G protein, Gor N protein of the rabies virus structural protein, herpes virus gDprotein, Japanese encephalitis virus E1 or pre-M protein, rotavirus coatprotein VP7 or coat protein VP4, human immunodeficiency virus gp120 orgp160 protein, Leishmania major surface antigen protein, malaria circumsporozoite major surface antigen protein, Toxoplasma 54-kd or CSprotein, cell surface protein PAc of caries-causing Streptococcusmutans; a gene encoding tumor regression antigens such as MAGE-1,MAGE-3, and BAGE, tissue-specific antigens such as tyrosinase, Mart-1,gp100 and gp75, p15, Muc1, CEA, HPV, E6, E7, HPR2/neu, etc.; and thegenes which are described in “Immunization with DNA”; Journal ofImmunological Methods, vol. 176, 1994, pages 145-152.

In the case that nucleic acids are oligonucleotides, they includes abase sequence, of which at least the portion binds complementarily underphysiological condition to a sense or antisense strand of a geneencoding a protein that has a physiological effect to disrupt thehomeostasis of a living body, a gene specific to pathogenic viruses,bacteria or the like, and, more specifically, they include a basesequence that binds complimentarily to the messenger RNA of a genespecific to a pathogenic virus, a bacterium or the like, or a geneencoding a protein having a physiological effect to disrupt thehomeostasis of a living body.

More specifically, oligonucleotides as used in the present inventionincludes a sequence complementary to a region containing an initiationcodon of a messenger RNA, or to a splicing site of a precursor messengerRNA. Examples of the oligonucleotides as used in the present inventioninclude, for example, those used for treatment or prevention of acancer, such as an oligonucleotide having a sequence of5′-CTCGTAGGCGTTGTAGTTGT-3′ (SEQ ID NO:1) which specifically inhibits theexpression of hst-1; ISIS3521 which specifically inhibits the expressionof protein kinase Cα to effectively treat progressive cancers such asnon-small cell lung carcinoma and colon cancer, and which has beenapplied in the trials to the treatment of prostate cancer, breastcancer, ovary cancer, pancreas cancer, large intestinal cancer, smallcell lung carcinoma; ISIS5132/CGP69846A which specifically inhibits theexpression of C-raf kinase and has been applied in the trials to thetreatment of prostate cancer, breast cancer, ovary cancer, cephalophyma,pancreas cancer, large intestinal cancer, small cell lung carcinoma;ISIS 2503 which specifically inhibits the expression of Ha-ras and hasbeen applied in the trials to the treatment of large intestinal cancer,breast cancer, cephalophyma, pancreas cancer, and small cell cancer;GEM231 which specifically inhibits the expression of protein kinase Atype I; MG98 which specifically inhibits the expression of DNA methyltransferase; INXC-6295 which inhibits the expression of c-myc; INX-3001which inhibits the expression of c-myb and has been considered to beapplied to the treatment of leukemia; G-3139 (Genasense) which inhibitsexpression of bc1-2 and has been considered to be applied to thetreatment of non-Hodgkin's lymphoma, large intestinal cancer, small celllung carcinoma, chronic lymphatic leukemia, acute myeloid leukemia,breast cancer, lymphoma, melanoma, myeloma, non-small cell lungcarcinoma, prostate cancer; an oligonucleotide which inhibits theexpression of MDM2 protein; an oligonucleotide which inhibits theexpression of VEGF and the like. Further, examples of theoligonucleotides used for treatment or prevention of infectious diseasesinclude GEM92 and GPI-2A which inhibits the growth of HIV. ISIS2922(fomivirsen), Vitravene, ISIS13312 and GEM132 which inhibits the growthof cytomegalovirus, ISIS14803 which inhibits the growth of hepatitis Cvirus, and the like. Examples of the oligonucleotides used for treatmentof inflammation include ISIS2302 which specifically inhibits theexpression of ICAM-1, and which has been applied in the trials to thetreatment of Crohn's disease, ulcerative colitis, kidney transplantationrejection inhibition, psoriasis, asthma; EPI-2000 which inhibits theexpression of adenosine A1 receptor and has been applied in the trialsto the treatment of asthma; and oligonucleotides which inhibit theexpression of TNF-α, CD49d (VLA-4), VCAM-1, PECAM-1 and the like.Further, examples of the oligonucleotides that prevent the restenosisafter percutaneous transluminal coronary angiogenesis include Resten-NGthat inhibits the expression of c-myc.

Genes encoding a protein that exhibits a physiological effect to disruptthe homeostasis include, for example, a series of genes, so-calledcancer genes. Specifically, they include genes for growth factors,receptor type tyrosine kinases, non-receptor type tyrosine kinases,GTP-binding proteins, serine-threonine kinases, transcription factorsand the like. More specifically, they include genes coding for hst-1 orornithine decarboxylase and the like.

The preparation for facilitating the transfer of a nucleic acidaccording to the present invention may further comprise apharmaceutically acceptable additive as appropriate in addition to thecomplex of the present invention. Pharmaceutically acceptable additivesinclude an agent making isotonic, a pH modifier, and a soothing agent incase of the use of the complex as injection, and an excipient, adisintegrator, and a coating agent in case of the use of the complex assolid, as well as those described in Japanese Handbook of PharmaceuticalExcipients (Japan Pharmaceutical Excipients Council). Specific examplesinclude salts and saccharides, which are used to keep the pH 6-8, or tomake isotonic with cells.

The preparation for facilitating the transfer of a nucleic acidaccording to the present invention may be in a form of solid orsolution. The preparation in a form of solid is loaded to a desired cellas it is, or after making a solution with a purified water, aphysiological solution, a buffer isotonic with living bodies, or thelike. Such preparation in a solution form also constitutes a part of thepresent invention.

The administration of the preparation for facilitating the transfer of anucleic acid according to the present invention may be selected fromoral route, injection, eye drop, nasal drop, transpulmonary route,transdermal absorption, and oral route and injection are preferred. Thepreparation may be administered to various sites depending on diseases,and may be placed on the necessary site at the time of operation.

In the present embodiment, the invention provides:

-   (1) A preparation for facilitating the transfer of a nucleic acid    into a target cell, which comprises as an essential component a    complex, preferably particles of a complex comprising a desired    nucleic acid and a collagen or a collagen derivative, wherein the    major axis of the particle is preferably 300 nm to 300 μm, more    preferably 300 nm to 100 μm, even more preferably 300 nm to 50 μm,    still more preferably 300 nm to 30 μm;-   (2) The preparation for facilitating the transfer of a nucleic acid    according to (1) wherein the target cell is an animal cell;-   (3) The preparation for facilitating the transfer of a nucleic acid    according to (2) wherein the target cell is an organ or a tissue    which requires to be treated, or its cell around;

(4) The preparation for facilitating the transfer of a nucleic acidaccording to any one of (1) to (3) wherein the collagen isatelocollagen:

(5) The preparation for facilitating the transfer of a nucleic acidaccording to any one of (1) to (4) wherein the molecular weight of thecollagen is from about 300,000 to about 900,000;

-   (6) The preparation for facilitating the transfer of a nucleic acid    according to any one of (1) to (5) wherein the collagen derivative    is a gelatin or a gelatin-crosslinking complex;-   (7) The preparation for facilitating the transfer of a nucleic acid    according to any one of (1) to (5) wherein the collagen derivative    is a collagen-crosslinking complex;-   (8) The preparation for facilitating the transfer of a nucleic acid    according to any one of (1) to (7) wherein the nucleic acid is an    oligonucleotide;-   (9) The preparation for facilitating the transfer of a nucleic acid    according to (8) wherein the oligonucleotide is from 5 to 30 mer in    length;-   (10) The preparation for facilitating the transfer of a nucleic acid    according to (8) or (9) wherein the oligonucleotide is a DNA or a    DNA derivative;-   (11) The preparation for facilitating the transfer of a nucleic acid    according to (8) or (9) wherein the oligonucleotide is an RNA or an    RNA derivative;-   (12) The preparation for facilitating the transfer of a nucleic acid    according to (10) or (11) wherein the DNA derivative or the RNA    derivative has at least one phosphorothioate bond;-   (13) The preparation for facilitating the transfer of a nucleic acid    according to any one of (1) to (7) wherein the nucleic acid is a    ribozyme or an oligonucleotide;-   (14) The preparation for facilitating the transfer of a nucleic acid    according to any one of (1) to (7) wherein the nucleic acid is a    plasmid DNA;-   (15) The preparation for facilitating the transfer of a nucleic acid    according to (13) wherein the plasmid DNA encodes a protein that    exhibits a physiological activity to treat or ameliorate    pathological conditions;-   (16) The preparation for facilitating the transfer of a nucleic acid    according to (13) wherein the plasmid DNA encodes a protein that    induces an immunological response to treat or ameliorate    pathological conditions;-   (17) The preparation for facilitating the transfer of a nucleic acid    according to (1) to (16) wherein the preparation is in a form of    solution, and the complex comprises 10 mg/ml or less of a nucleic    acid;-   (18) The preparation for facilitating the transfer of a nucleic acid    according to (17) wherein the complex comprises 1 mg/ml or less of a    nucleic acid;-   (19) The preparation for facilitating the transfer of a nucleic acid    according to (18) wherein the complex comprises 500 μg/ml or less of    a nucleic acid;-   (20) The preparation for facilitating the transfer of a nucleic acid    according to (1) to (19) wherein the preparation has pH5 to pH9,    preferably, pH6 to pH8;-   (21) The preparation for facilitating the transfer of a nucleic acid    according to (1) to (20) further comprising phosphoric acid in a    concentration from 0.001M to 0.1M;-   (22) The preparation for facilitating the transfer of a nucleic acid    according to (21) further comprising phosphoric acid in a    concentration from 0.01M to 0.1M;-   (23) The preparation for facilitating the transfer of a nucleic acid    according to (1) to (22) further comprising an agent that inhibits    the formation of collagen association body, for example sucrose or    arginine; and-   (24) The preparation for facilitating the transfer of a nucleic acid    according to (1) to (24) further comprising an appropriate amount of    a pharmaceutically acceptable additive.

3) Complexes

As the third embodiment, the present invention provides a complexcomprising a collagen or a collagen derivative and a desired nucleicacid. The complexes include electrostatic complexes that are bound andformed by the electrostatic force, and physical complexes that are boundand formed by the physical force such as hydrophobic binding. Bothbinding systems may coexist, and this embodiment is also fallen in thescope of the present invention.

It has been found for the first time that a collagen iselectrostatically interacted with a nucleic acid to form a complex.

As used herein, “electrostatic complexes” means polyionic complexesbetween a collagen or a collagen derivative having many electric chargesin the molecule and a nucleic acid, and specifically means bindingbodies wherein a collagen or a collagen positively charged electricallyattract to a nucleic acid negatively charged. In the polyioniccomplexes, many counter ions are released from the molecule on thecomplex formation, and therefore very large increase in entropy isproduced. In view of the formation of such electrostatic complexes, itis understood that the sustained expression of a nucleic acid asobserved in the working examples would result from the complex formationleading to the increased stability of nucleic acids within cells.

Minimum unit of the complex of the present invention is a complex formedby one molecule of collagen and one molecule of nucleic acid. We foundthat a collagen forms a complex with a nucleic acid, and stimulatinglyassociates with another collagen to form an association body. Theassociation body is formed in a manner that a collagen molecule having acylindrical shape wherein the major axis is about 300 nm and thediameter is 1.5 nm is predominantly associated parallel to thelongitudinal axis of the molecule. Accordingly, the complexes are formedby the noncovalent binding between many association bodies and manynucleic acid molecules, and include filamentous complexes having alongitudinal axis of 1 nm or more as a result of the largely developedextension of the bodies, filamentous complexes wherein fine bodies areeach bound via nucleic acids, and particulate complexes formed by morefine complexes and nucleic acids.

The complexes of the present invention can be various in shape.

The complexes of the present invention are preferably in a form ofparticle. As used herein “particle” means a shape that a collagen or acollagen derivative could assume, and does not necessarily mean aspherical shape. Minimum size of the complexes of the present inventionis 300 nm that corresponds to the major axis of a complex formed by onemolecule of collagen. The particles have a major axis of 300 nm to 1 mm,and in view of transfer efficiency of nucleic acids, they preferablyhave a major axis of 300 nm to 300 μm, more preferably 300 nm to 100 μm,even more preferably 300 nm to 50 μm, still even more preferably 300 nmto 30 μm.

We found that the ratio of a collagen or a collagen derivative to anucleic acid composed in a complex is responsible for the fact that thecomplex can assume various forms or shapes, and that the form of thecomplex is dependent exclusively on the development of the association(fibrosis) of the collagen or collagen derivative promoted by theformation of the complex with the nucleic acid. We also found that theexcess formation of the association bodies is not suitable for thetransfer of the nucleic acid, and found that the form or the shape ofthe complex could be controlled by adjusting the concentration of thecollagen or collagen derivative and the desired nucleic acid to bemixed, as well as environmental factors such as salt concentration,temperature, pH, and glucose concentration. Further, we observed that aplasmid DNA having 1000 by or more is extremely promoted in theassociation, whereas a plasmid DNA having a 100 by or less is weaklypromoted in the association, and found that the association of collagenor collagen derivative involved in the complex formation is affected bythe length of the nucleic acid, concluding that there is a compositionof a collagen or collagen derivative and a nucleic acid optimal for theassociation depending on the length of the nucleic acid.

The form of the shape of the complex may affect the transfer efficiencyof nucleic acids into cells. The finding that collagens are complexedwith nucleic acids, and the shape of the complex affects the transferefficiency and the expression efficiency of nucleic acids in cellsprovides a strategy for optimizing the transfer efficiency of nucleicacids. Liposomes as used in laboratory to transfer nucleic acids intotarget cells are difficult to be widely utilized from practical viewpoints, since they tend to aggregate together immediately when combinedwith nucleic acids, are varied in transfer efficiency on use, and arenecessarily prepared just before use. However, the complexes of thepresent invention are stable in the form or the shape when stored incold space. The most important issue in gene transfer technique is thatwidely used methods are quite few. The complexes of the presentinvention are stable in the form or the shape and could be usedpractically.

In the present embodiment, the invention provides:

-   (1) A particle of the complex comprising a desired nucleic acid and    a collagen or a collagen derivative;-   (2) The particle of the complex according to (1), wherein the major    axis is 300 nm to 300 μm;-   (3) The particle of the complex according to (2), wherein the major    axis is 300 nm to 100 μm;-   (4) The particle of the complex according to (3), wherein the major    axis is 300 nm to 50 μm, preferably 300 nm to 30 μm;-   (5) The particle of the complex according to (1) to (4), wherein the    nucleic acid is an plasmid DNA;-   (6) The particle of the complex according to (5), wherein the ratio    of the number of a collagen molecule or a collagen derivative    molecule to the number of a nucleotide monomer of the plasmid DNA is    1:20 to 1: the number of a nucleotide monomer of the plasmid DNA,    preferably 1:50 to 1: the number of a nucleotide monomer of the    plasmid DNA, more preferably 1:50 to 1:4000, even more preferably    1:50 to 1:2000, still even more preferably 1:50 to 1:1000;-   (7) The particle of the complex according to (6), wherein the ratio    is 1:96 to 1: the number of a nucleotide monomer of the plasmid DNA;-   (8) The particle of the complex according to (7), wherein the ratio    is 1:96 to 1:1122;-   (9) The particle of the complex according to (8), wherein the ratio    is 1:96 to 1:701;-   (10) The particle of the complex according to (1) to (4), wherein    the nucleic acid is an oligonucleotide;-   (11) The particle of the complex according to (10), wherein the    ratio of the number of a collagen or a collagen derivative to the    number of a nucleotide monomer of the oligonucleotide in the complex    is 1:1 to 1:200, preferably 1:3 to 1:150, more preferably 1:3 to    1:120;-   (12) The particle of the complex according to (11), wherein the    ratio is 1:20 to 1:120;-   (13) The particle of the complex according to (12), wherein the    ratio is 1:50 to 1:120;-   (14) The particle of the complex according to (1) to (13), which is    comprised in a solution of pH 5 to pH 9, preferably pH 6 to pH 8;-   (15) A process for preparing a particle of the complex according    to (1) to (13), which comprises mixing a collagen or a collagen    derivative and a desired nucleic acid in a solution cooled to 10    ° C. or less;-   (16) A process for preparing a particle of the complex according    to (1) to (13), which comprises mixing a collagen or a collagen    derivative and a desired nucleic acid in a solution comprising    phosphoric acid in a concentration from 0.001M to 0.1M;-   (17) A process for preparing a particle of the complex according    to (1) to (13), which comprises mixing a collagen or a collagen    derivative and a desired nucleic acid in a solution comprising    phosphoric acid in a concentration from 0.01M to 0.1M;-   (18) A process for preparing a particle of the complex according    to (1) to (13), which comprises mixing a collagen or a collagen    derivative and a desired nucleic acid in a solution comprising an    agent that inhibits the formation of collagen association body, for    example sucrose or arginine;-   (19) A process for preparing a particle of the complex according    to (1) to (13), which comprises mixing a collagen or a collagen    derivative and a desired nucleic acid in a solution comprising an    appropriate amount of a pharmaceutically acceptable additive wherein    the pharmaceutically acceptable additive is as defined above;-   (20) A process for preparing a particle of the complex according    to (1) to (13), which comprises combining two or more of the    processes according to (14) to (19);-   (21) A process for preparing a particle of the complex according    to (1) to (13), which further comprises penetrating the complex    through a filter having a pore size of 100 μm or less for size    selection.-   (22) The process according to (21), which comprises penetrating the    complex through a filter having a pore size of 10 μm or less for    size selection;-   (23) A process for preparing a particle of the complex according    to (1) to (13), which further comprises centrifuging the complex at    10,000 rpm or more for concentration and isolation;-   (24) The process according to (23), which comprises centrifuging the    complex at 50,000 rpm or more.

As used herein, “the number of a nucleotide monomer” means the number ofnucleotide monomer units composing of a desired nucleic acid. As usedherein, “the ratio of the number of a collagen molecule or a collagenderivative molecule to the number of a nucleotide monomer” means thenumber of nucleotide monomers in a desired nucleic acid relative to onemolecule of a collagen or collagen derivative comprising anelectrostatically or physically bound complex. The number of anucleotide monomers is almost the same as “negative charge of a desirednucleic acid”. As used herein, “negative charge of a desired nucleicacid” means the number of phosphate groups intervening betweennucleotides composing of a nucleic acid. The numeral values of thenegative charge of a nucleic acid is equal to the number of phosphategroups of the nucleic acid, phosphate groups intervening betweennucleotides, and phosphorus-containing groups intervening betweennucleotides (phosphate groups and phosphorothioate groups).

As used herein, “an agent that inhibits the formation of collagenassociation body” includes an agent that inhibits electrostaticinteractions that would cause the formation of collagen association bodyvia charges of basic and acidic amino acids comprised mainly in acollagen molecule, and an agent that inhibits the ordered arrangement ofcollagen molecules. Examples of the former include salts amino acids,and urea, and examples of the latter include saccharides such assucrose, and arginine.

The particle of the complex according to the invention may be comprisedin the preparation for facilitating the transfer of a nucleic acidaccording to the invention.

4) Medical Instruments and Cell Culture Instruments

As the fourth embodiment, the invention provides a medical instrument ora cell culture instrument, of which the surface is coated with aparticle of the complex according to the present invention.

It has been found that the transfer efficiency of a nucleic acid isimproved when a particle of the complex according to the presentinvention is coated onto a solid surface, and target cells are contactedthereto, compared to dropwise addition of a particle of the complexaccording to the present invention to the target cells, in other words,solid coating of the complex particles is superior to dropwise additionin terms of transfer efficiency (as shown in Example 6).

Specifically, medical instruments and cell culture instruments accordingto the present embodiment include artificial vascular grafts, medicalstents, and artificial hearts. Artificial vascular grafts are requiredto allow vascular endothelial cells to proliferate and spread in theirinner side in order to inhibit fibrin development and suppress thecomplement activation within the vessels. Thus, when coated ontoartificial vascular grafts, a particle of the complex according to thepresent invention wherein nucleic acids encoding endothelial growthfactors such as a vascular endothelial growth factor (VEGF) are bound toa collagen is expected to readily and rapidly proliferate vascularendothelial cells.

Cell culture instruments, of which the surface is coated with a particleof the complex comprising a desired nucleic acid and a collagen or acollagen derivative include plates, flasks, 96-well microplates asusually used in cell culture experiments. Example 6 hereinafterdemonstrated that amounts of the complex particles coated onto the solidsurface per unit area drastically affect transfer efficiency of nucleicacids into cells. Accordingly, the amounts of the complex particlescoated onto the solid surface per unit area constitute a part of thepresent invention.

In the present embodiment, the invention specifically provides:

-   (1) A medical instrument or a cell culture instrument that is coated    with a particle of the complex comprising a desired nucleic acid and    a collagen or a collagen derivative;-   (2) The medical instrument or the cell culture instrument according    to (1), wherein the particle of the complex is coated so that an    amount from 0.1 μg to 50 μg of a nucleic acid is comprised in 1    square centimeter;-   (3) The medical instrument or the cell culture instrument according    to (1), wherein the particle of the complex is coated so that an    amount from 0.1 μg to 50 μg of a nucleic acid is comprised in 1    square centimeter;-   (4) The medical instrument or the cell culture instrument according    to (3), wherein the particle of the complex is coated so that an    amount from 1 μg to 10 μg of a nucleic acid is comprised in 1 square    centimeter;-   (5) The medical instrument or the cell culture instrument according    to (1), wherein the particle of the complex having a major axis of    300 nm to 300 μm is released from the solid surface when exposed in    a solution isotonic with a living body; and-   (6) The medical instrument or the cell culture instrument according    to (5), wherein the solution isotonic with a living body is a    phosphate buffer comprising sodium chloride.

Feasible distribution of the invention on the market is important tocarry out the present embodiment. As described above, liposomes arerequired to be prepared just before use, meaning extremely poordistribution. In general, it is believed difficult to distributeadenovirus as coated onto the solid surface according to the presentembodiment. However, it has been found unexpectedly that adenovirus ascoated and dried on solid surface together with a collagen according tothe present invention retain the infectivity at room temperature for 7days. This shows that the medical instrument and the cell cultureinstrument according to the invention could be feasibly distributed onthe market.

5) A Process for Transferring a Desired Nucleic Acid into a Target Cellor a Process for Improving the Expression Level of a Desired NucleicAcid in a Target Cell

As described above, a particle of the complex according to the presentinvention can be used to facilitate the transfer of a desired nucleicacid into a target cell. Thus, as the fifth embodiment, the presentinvention provides a process for transferring a desired nucleic acidinto a target cell or a process for improving the expression level of adesired nucleic acid in a target cell, which comprises using a particleof the complex according to the present invention. As used herein,“improving the expression level” means the increasing of the expressionlevel of a desired nucleic acid or the extension of the duration time ofthe expression.

Thus, in the present embodiment, the invention provides:

-   (1) A process for transferring a desired nucleic acid into a target    cell, which comprises using a particle of the complex as defined    above comprising a desired nucleic acid and a collagen or a collagen    derivative;-   (2) A process for improving the expression level of a desired    nucleic acid in a target cell, which comprises using a particle of    the complex as defined above comprising a desired nucleic acid and a    collagen or a collagen derivative;-   (3) The process according to (2), wherein the process is to improve    the expression level, or to extend the duration time of the    expression;-   (4) The process according to any one of (1) to (3), wherein the    particle of the complex as defined above is coated onto the solid    surface, and the target cell is cultured on the solid surface.-   6) A process for examining the function of a gene or a protein in a    target cell

According to the present invention wherein the transfer of a nucleicacid into a cell is facilitated, it is possible to readily examine thefunction of a gene or a protein in a target cell. For example, humangenome project identify a large number of genes, and showed the basesequences thereof. It is necessary to clarify the functions ofidentified genes in order to utilize those information practically inthe field of medication or food industry. However, it has been clearthat the conventional approach to examine the function by producing andpurifying proteins from genes one by one is time-consuming and notpractical. Accordingly, a process for examining the function of a genewhich comprises transferring and expressing a plasmid DNA incorporatedwith a gene to be examined into a cell, or a process for examining thefunction of a gene which comprises transferring an antisenseoligonucleotide that inhibits the expression of a gene to be examinedinto a cell and inhibiting the gene expression should be useful. In caseof the examination of the gene function by phenotype of the cell asshown above, it is necessary to conduct the process for transferring aplasmid DNA or an antisense DNA into a cell without adverse affection tothe cell as much as possible. Thus, a liposome, which is high incytotoxicity, would affect the information as obtained due to itscytotoxicity. On the other hand, a collagen as used herein hardly affectthe cells since a collagen originally exist in a living body andcontacts with the cells, and therefore the invention enables themeasurement of gene functions without noise. Particular measurementscomprise mixing a plasmid DNA or an adenovirus expressing a gene to beexamined, or an antisense oligonucleotide inhibiting the expression of agene to be examined, allowing the formation of particles of the complex,then coating and arranging the same on the solid surface of the cultureplate. Solid plates as used herein include 96-well multiwellplates andmicroplates. After the coated complex particles are dried andimmobilized on the solid, cells are seeded and cultured on the plate forseveral days. The coated complex particles are transferred efficientlyinto cells attached to the coated part, and allow the expression of agene to be examined or the inhibition of the same for a long period oftime. After a few days, the functions of target genes can be clarifiedby examining the morphology of the cells, the level of the geneexpression in the cells, or the kinds or the amounts of the proteinsproduced by the cells. The features of the present invention alsoinclude selective and efficient transfer of the complex particles coatedonto the solid into the cells attached to the coated part. In otherwords, it is possible to examine the functions of a large number ofgenes on microplates at a time without computing of the cells by wells.

In the present embodiment, the invention provides:

-   (1) A process for examining the function of a gene or a protein in a    target cell, which comprises coating a solid surface with a particle    of the complex according to above (3) of the present invention that    comprises the gene, a gene encoding the protein, or a nucleic acid    inhibiting the expression of the gene or the protein in a cell;    culturing the target cell on the solid surface; and examining the    expression level of the nucleic acid or the expression level of the    gene or the protein in the target cell, or the proliferation ratio    or the phenotype of the cell;-   (2) The process according to (1), wherein the gene or a nucleic acid    encoding the protein is a plasmid DNA, and the expression level of    the nucleic acid is examined; and-   (3) The process according to (1), wherein the gene or a nucleic acid    that inhibits the expression of the protein in a cell is an    antisense oligonucleotide or a ribozyme, and the expression level of    the gene or the protein is examined.-   (7) A process for screening for a nucleic acid that treats a disease

According to the present invention wherein the transfer of a nucleicacid into a cell is facilitated, it is possible to screening for anucleic acid that is capable to compensate normal genes, repair orcorrect defective genes so as to treat various diseases such as geneticdiseases, cancers, AIDS, rheumatoid arthritis, lifestyle-relateddiseases. For example, after the complex particles are formed with anucleic acid that is examined for a therapeutic effect on diseases and acollagen, and are coated, dried and immobilized on the solid asdescribed in 6) above, cells presenting pathological condition arecultured on the plate, transferring efficiently the nucleic acid intothe cell presenting pathological condition. Effects of the nucleic acidmay be examined on the basis of change in the morphology of the cells,cell death, cell proliferation, pattern of the gene expression in thecells, or the kinds or the amounts of the proteins produced by thecells. It is evident that, in the nucleic acid transfer in theembodiment, affection by vectors to be transferred should be minimized,and a collagen as used herein makes it possible to examine the effect onpathological condition without noise. The features of the presentinvention also include coincidental examinations of a large number ofgenes, as shown by the fact that nucleic acids that is to be examinedfor function can be immobilized and arranged on a cell culture solidcarrier having a tiny area.

In the present embodiment, the invention provides:

-   (1) A process for screening for a nucleic acid that treats a    disease, which comprises coating a solid surface with a particle of    the complex according to the present invention that comprises a    nucleic acid candidate that inhibits the expression of a gene    associated with the disease in a cell; culturing the cell presenting    the condition of the disease on the solid surface; and examining the    expression level of the gene to be inhibited with each of the    nucleic acid candidate, or the proliferation ratio or the phenotype    of the cell;-   (2) A process for examining a therapeutic effect of a nucleic acid    to be expected to inhibit the expression of a gene associated with a    disease, which comprises coating a solid surface with a particle of    the complex according to the present invention that comprises the    nucleic acid; culturing the cell presenting the condition of the    disease on the solid surface; and examining the expression level of    the gene, or the proliferation ratio or the phenotype of the cell;-   (3) The process according to (1) or (2), wherein the nucleic acid is    to inhibit the expression of the gene associated with the disease in    the cell, or to have a function that inhibits the expression of the    gene associated with the disease in the cell; and-   (4) The process according to (3), wherein the nucleic acid that    inhibits the expression of the gene associated with the disease in    the cell is a plasmid DNA encoding the gene, or the nucleic acid    that have a function that inhibits the expression of the gene    associated with the disease in the cell is an antisense    oligonucleotide or a ribozyme.

8) Other Embodiments

In the present embodiments, the invention provides:

-   (1) A cell culture instrument, of which the surface is coated with a    desired nucleic acid together with a collagen or a collagen    derivative; preferably the cell culture instrument wherein the    desired nucleic acid is complexed with the collagen or the collagen    derivative to form a particle of the complex;-   (2) A cell culture instrument, of which the surface is coated with a    film comprising a collagen or a collagen derivative containing a    desired nucleic acid; preferably the cell culture instrument wherein    the desired nucleic acid is complexed with the collagen or the    collagen derivative to form a particle of the complex;-   (3) A cell culture instrument, on which the surface is coated with a    film comprising a collagen or a collagen derivative, and a desired    nucleic acid;-   (4) The cell culture instrument according to (1) to (3), wherein the    nucleic acid constitutes a library;-   (5) The cell culture instrument according to (4), wherein the    nucleic acid is a cDNA or oligonucleotide that constitutes a    library;-   (6) The cell culture instrument according to (4) or (5), wherein the    nucleic acid that constitutes a library is comparted each other at a    distance;-   (7) A film comprising a collagen or a collagen derivative and a    desired nucleic acid wherein the nucleic acid that constitutes a    library is comparted each other at a distance; preferably the film    wherein the nucleic acid is complexed with the collagen or the    collagen derivative to form a particle of the complex;-   (8) The film according to (7), wherein the nucleic acid is a cDNA or    oligonucleotide that constitutes a library;-   (9) The cell culture instrument according to (2) or (3), on which    the surface is coated with the film according to (7) or (8);-   (10) The cell culture instrument according to (9), that is used for    the expression of proteins;-   (11) The cell culture instrument according to (9), that is used for    the inhibition of gene expression;-   (12) A process for examining the function of a gene in a cell, which    comprises culturing the cell on the cell culture instrument    according to (1) to (6) on which a nucleic acid containing a cDNA of    the gene is immobilized; and examining the proliferation ratio or    the phenotype of the cell, or the production level of certain    proteins;-   (13) A process for examining the function of a gene in a cell, which    comprises culturing the cell on the cell culture instrument    according to (1) to (6) on which an oligonucleotide containing a    base sequence complementary to a messenger RNA of the gene is    immobilized; and examining the proliferation ratio or the phenotype    of the cell, or the production level of certain proteins; and-   (14) The cell culture instrument according to (1) to (6), wherein    the surface of the cell culture instrument outside the part    immobilized by the nucleic acid is hydrophilic or hydrophobic as    high as the cell is not attached;

In order to transfer a nucleic acid into a target cell on solid phase,the cell may be cultured on a cell culture instrument, on which adesired nucleic acid together with a collagen or a collagen derivative,or a desired nucleic acid complexed with the collagen or the collagenderivative to form a particle of the complex is directly immobilized.Alternatively, target cells may be cultured on a cell cultureinstrument, of which the surface is covered with a film comprising acollagen or a collagen derivative containing a desired nucleic acid, ora film comprising a particle of the complex, and a film made of agaroseand albumin comprising a collagen or a collagen derivative containing adesired nucleic acid, or a particle of the complex may be used.

The present invention comprises making a library that is constitutedwith various nucleic acids complexed with a collagen or a collagenderivative independently on the solid phase, and makes it possible toexamine the functions of the gene in cells at the same time and in thesame condition. Further, the invention provide a library for geneexpression or a library for inhibition of gene expression wherein anucleic acid or an oligonucleotide comprising the libraryed cDNA isarranged on the solid phase, allowing the examination of the genefunctions at a stage of cell. Nucleic acids comprising the cDNA thatconstitutes a library are not limited to a specific species, and includeGene Storm pcDNA3.1 vector (In Vitrogen, Inc.).

When a complex comprising a nucleic acid is arranged on the solid phase,and cells are cultured thereon to examine the functions of thetransferred gene, it is necessary to avoid the contamination of thecells, each of which is transferred with respective nucleic acidindependently arranged.

The features of the present invention also include selective andefficient transfer of the complex particles coated onto the solid intothe cells attached to the coated part. In other words, it is possible toexamine the functions of a large number of genes on microplates at atime without computing of the cells by wells. To do so, wells may beused to compart each part arranged by the nucleic acids, and 6 to 384wells may compart one plate. In addition to the wells, alternatively,surface on the solid phase that is hydrophilic or hydrophobic as high asthe cell is not attached may be used to prevent the cells from movingacross the parts arranged by the nucleic acids. As used herein, thesurface that is highly hydrophilic means surface having a water-contactangle of 40 degree or less, and the surface that is highly hydrophobicmeans surface having a water-contact angle of 110 degree or more.Distance between the arranged nucleic acids should be kept over thelength of the most extension of the seeded cells. Thus, when the surfaceof the cell culture instrument outside the part immobilized by thenucleic acid or the part coated with a film comprising a nucleic acid,it is not necessary to carry out culture by computing the cells withwells, and it is possible to examine the functions of a large number ofthe genes on a microplate.

Examples

The present invention is further illustrated by the following Examplesand Experiments, but is not restricted by these Examples and Experimentsin any way.

Example 1 Formation of Complexes Between a Plasmid DNA and a Collagen

Both equal amounts of an aqueous solution containing a plasmid DNA(pCAHST-1: 7.9 Kbp) incorporated with a gene of fibroblast growth factorHST-1 (FGF4) gene (Proc. Natl. Acad. Sci. USA, 84, 2890-2984 (1987)) at10 μg/ml, and of a neutral aqueous solution containing 200, 60, 20, 6,2, 0.6, and 0 μg of an atelocollagen (KOKEN CO., LTD.) were mixedtogether, and the mixtures were analyzed on agarose gel electrophoresis.Agarose gel electrophoresis was carried out with 0.8% agarose gel in TAE(Tris-acetate) buffer at Horizontal Electrophoresis Unit (Mupid, AdvanceCO.). After electrophoresis, the gel was stained with ethidium bromide,and photographed with a transilluminator. The results are shown inFIG. 1. pCAHST-1 in the presence of the collagen at 10 μg/ml or more didnot electrophorese, and retained on the well. This means that pCAHST-1at 5 μg/ml was complexed with the collagen at 10 μg/ml.

Similarly, both equal amounts of 10 μg/ml pCAHST-1 and 100 μg/mlcollagen were mixed together in 10 mM Tris hydrochloride buffer (pH7.5)containing 1.0 M sodium chloride, and the mixture was electrophoresed,which results are shown in FIG. 2. When 1.0 M sodium chloride coexisted,pCAHST-1 irrespective of the presence of the collagen at 100 μg/ml didelectrophorese. This means that a complex formed by pCAHST-1 and thecollagen should be an electrostatic one.

Further, the formation of a complex between 10 μg/ml pCAHST-1 and thecollagen in 10 mM Tris hydrochloride buffer (pH7.5) containing 1.0 Msodium chloride was compared between the presence and the absence ofheparan sulfate. The results are shown in FIG. 3. In the absence ofheparan sulfate, all of 5 μg/ml pCAHST-1 were complexed with 100 μg/mlcollagen, whereas in the presence of heparan sulfate, it found thatthere were some pCAHST-1 that were not complexed with collagen even at300 μg/ml. This means that heparan sulfate having a negative chargesimilar to pCAHST-1 cause the competition in complex formation with thecollagen between pCAHST-1 and heparan sulfate, suggesting that pCAHST-1and a collagen form a electrostatic complex.

Example 2 Microscopy of Complexes

Both equal amounts of an aqueous solution containing pCAHST-1 at 200μg/ml, and an aqueous solution containing an atelocollagen at 0, 20,200, and 1000 μg/ml were mixed, and the mixture was added with PicoGreendsDNA Quantitation Reagent (Molecular Probes) to stain pCAHST-1 andobserved by microscopy. The results are shown in FIG. 4. In case thatthe collagen concentration after the mixing is 500 μg/ml (0.05% collagenin the figure), the filamentous complexes having a major axis of morethan 1 mm were predominantly observed, whereas the particulate complexeshaving a major axis of 10 to 100 μm were predominantly observed in casethat the collagen concentration is 100 μg/ml (0.01% collagen in thefigure), and the particulate complexes having a major axis of 10 μm orless were predominantly observed in case that the collagen concentrationis 10 μg/ml (0.001% collagen in the figure). This means that theformation of the complexes can be controlled by the concentrations ofplasmid DNAs and collagens at the time of mixing. These complexesmaintained stably their shape over a week or more when stored at 5° C.(FIG. 5). This means that the complexes can be stored for a long periodof time, and be distributed on the market at they are, not requiring thepreparation just before use.

Example 3 Extension Effect of Complexes on the Expression Duration Time

Both equal amounts of an aqueous solution containing a plasmid DNAencoding a fluorescent protein (EGFP) (pCMV-EGFP/pEGFP-N1, Clontech Co.)at 200 μg/ml, and an aqueous solution containing an atelocollagen at0.02%(w/w) were mixed together to prepare a formulation in a gel form.

To human embryonic kidney cells, 293 cells cultured on a dish (diameter:6 cm), 100 μl of the gel formulation (containing 10 μg of pCMV-EGFP) wasadded in the presence of 2 ml of serum-free medium. Then, after culturedat 37° C. overnight, the cells were washed with PBS to remove theserum-free medium and the formulation, and cultured in a mediumcontaining 10% calf serum. The cells were observed by fluorescencemicroscopy with time course to check the EGFP expression. As a control,a PBS solution containing an equal amount of pCMV-EGFP, and a complex ofa cationic liposome formulation and pCMV-EGFP were each added to the 293cells.

The results are shown in FIG. 6. In case of the gel formation ofpCMV-EGFP, the expression of EGFP was observed, and the expression wasfound to maintain during a long period of time up to 52 days after theaddition. On the other hand, in case of the PBS solution containingpCMV-EGFP, no expression of EGFP was observed, and in case of thecationic liposome formulation, the expression of EGFP was found tomerely maintain during a short period of time for 2 days. These resultsshows that the formulation of a plasmid DNA with a collagen promotes theexpression efficiency of a plasmid DNA, and maintains the expressionduring a longer period of time.

This means that a complex as formed with a plasmid DNA and a collagenpromotes the transfer of a plasmid DNA into a cell, and enhances thestability of the plasmid DNA inside and outside the cell, resulting inextension of the gene expression in the cell. Without being limited to aparticular theory of operation, it is believed that the fact that theexpression was extended for a longer period of time in the absence ofany complex in the system shows that the complexes interact with thecells as strongly as that the complexes are not removed by the washing,or that the complexes are incorporated into the cells.

Example 4 Effect of the Composition of Complexes on Transfer Efficiencyof Plasmid DNAs

Using pCMV-EGFP as a plasmid DNA, complexes having the composition asshown in Table 1 were prepared.

TABLE 1 complex atelocollagen (%) pDNA (μg/ml) EGFG-1 0 100 EGFG-2 0.001100 EGFG-3 0.01 100 EGFG-4 0.05 100 EGFG-5 0.1 100

The results show that their transfer efficiencies are promoted in orderof EGFG-3 complex>EGFG-2 complex>EGFG-4 complex=EGFG-5 complex. Notransfer was found in EGFG-1. This shows that the formation of a plasmidDNA with a atelocollagen promotes the expression efficiency of a plasmidDNA, and that the expression efficiency in the complexes containing theequal amount of plasmid DNA varies depending on the content ofatelocollagen.

Example 5

Effects of the Amounts of Plasmid DNA on the transfer Efficiency of DNAPlasmid.

Zero, 10, 50, 100, 250, and 500 μl of EGFG-3 complex (0.01%atelocollagen, 100 μm/ml pCMV-EGFP), which was found in Example 4 thatthe transfer efficiency is the highest, was added to the 293 cellscultured on a 6-well dish in the presence of 1 ml of serum-free medium.Then, after cultured at 37° C. overnight, the cells were washed with PBSto remove the serum-free medium and the complex, and the medium wasreplaced for a 10% FBS (a medium containing 10% calf serum). The cellswere observed by fluorescence microscopy with time course over 6 days tocheck the EGFP expression.

When the amount of plasmid DNA is increased, then the transferefficiency was also improved. Particular data are shown in thefollowings. In the table, expression efficiency was estimated bycounting the number of the cells emitting fluorescence caused by EGFPexpression among the counted number of all cells existing in thecompartment defined by the microscopic range.

TABLE 2 Transfection efficiency EGFG-3 (μl) 100 250 500 EGFP expressingcells 262 1057 1081 (cells/wel) efficiency 0.006 0.03 0.03

Example 6 Comparison in Extension Effects of Complexes on Duration Timeof Expression Between the Dropwise Addition and Solid Coating of theComplexes

First, the composition of a collagen and a plasmid DNA was optimized onthe basis of the composition of EGFG-3 complex (0.01% atelocollagen, 100μg/ml pCMV-EGFP), which was found that the transfer efficiency is thehighest, and then, the duration times of expression were comparedbetween the dropwise addition and the solid coating.

-   (1) Using pCMV-EGFP as a plasmid DNA, complexes having the various    compositions of atelocollagen concentrations as shown in Table 3    were prepared.

TABLE 3 Experiment 6: Gene-transfection efficiency relative toatelocollagen complex atelocollagen (%) pDNA (μg/ml) EGFG-3A 0.005 100EGFG-3B 0.008 100 EGFG-3 0.01 100 EGFG-3C 0.015 100 EGFG-3D 0.02 100EGFG-3E 0.03 100 pDNA solely 0 100

The complexes prepared were added dropwise to the 293 cells cultured ona 6-well dish in the presence of serum-free medium. Then, after culturedat 37° C. overnight, the cells were washed with PBS to remove theserum-free medium and the complex, and the medium was replaced for amedium containing 10% calf serum. Seven days later, the cells wereobserved by fluorescence microscopy, and the cells expressing EGFP werecounted. The results are shown in FIG. 7. For EGFG-3B, 3D, and 3E,fluorescence intensities of the expressed EGFP were measured (Array ScanII System, Cellomics, Co.). Relative fluorescence intensities are shownin FIG. 8.

The complexes prepared were coated onto a 6-well dish at 250 μl/well,and the dish was air-dried for 30 minutes. Then, the 293 cells (4-5x10⁵cells/well) in a medium containing 10% FBS were added thereto, andcultured at 37° C. overnight. Seven days later, the cells were observedby fluorescence microscopy, and the cells expressing EGFP were counted.The results are shown in FIG. 9.

FIG. 7 showing the results of dropwise addition and FIG. 9 showing theresults of solid coating indicate that the concentration of collagenaffects the transfer efficiency, and the solid coating is superior tothe dropwise addition in transfer efficiency.

-   (2) Complexes having the various compositions of plasmid DNA    concentrations as shown in Table 4 were prepared, and transfer    efficiencies were compared between dropwise addition and solid    coating.

TABLE 4 complex atelocollagen (%) pDNA (μg/ml) EGFG-3B 0.008 100EGFG-3B2 0.008 60 EGFG-3B3 0.008 40 EGFG-3B4 0.008 20

The results of dropwise addition are shown in FIG. 10, and the resultsof solid coating are shown in FIG. 11. Relative fluorescence intensitiesof EGFP in each sample obtained in solid coating are also shown in FIG.12. These results show that the concentration of plasmid DNA affects thetransfer efficiency, specifically the expression level of EGFP and theconcentration of plasmid or DNA are in a positive correlation, and alsothat the solid coating is superior to the dropwise addition in transferefficiency.

Example 7 Application of Solid Coating to Screening Method

Ten μM of a phosphorothioate antisense oligonucleotide(5′-CTCGTAGGCGTTGTAGTTGT-3′; Molecular weight, about 6500; SEQ ID NO: 2)(5) (Sawaday) having a sequence complementary to a sequence from 4196 byto 4216 by of fibroblast growth factor HST-1 (FGF4) gene (described inProc. Natl. Acad. Sci. USA, 84, 2890-2984 (1987)) was mixed with a 0.05%solution of atelocollagen to form complexes. The complexes were coatedonto the bottom of a 96-well plate for cell culture, and air-dried toobtain a cell culture instrument wherein the bottoms were coated withthe complex. Similarly, a phosphorothioate sense oligonucleotide(5′-GAGCATCCGCAACATCAACA-3′; SEQ ID NO: 3) (1) having the same sequenceas the HST-1 gene, as well as phosphorothioate antisenseoligonucleotides having a sequence (5′-AGTCGCATGCACACAACACA-3′; SEQ IDNO: 4) (2) as obtained by scrambling the antisense oligonucleotidesequence, and the three random sequences (5′-GACCATCGTCGATTCCAGT-3′; SEQID NO: 5) (3), (5′-CATGAACATCCTGAGCATCC-3′; SEQ ID NO: 6) (4), and(5′-GTTCACGAAGAAAGAAGGCT-3′; SEQ ID NO: 7) (6)) were each complexed witha collagen to form complexes, which complexes were coated onto thebottom, and dried. Onto the plate coated with those oligonucleotides,NEC8 cells in which the proliferation was promoted by overexpression ofHST-1, and HepG2 cells in which the proliferation was promoted by thegene other than HST-1 were seeded at 0.5×10⁵ cells/well, and the cellswere cultured for 4 days. After the culture, the inhibition of cellproliferation was assayed using TetraColor ONE Cell Proliferation AssayReagent. Specifically, the sample solution stained was determinedspectrometrically at 650 nm, using the absorbance at 450 nm as control(FIG. 13).

As a result, the inhibitory effect of NEC 8 cells proliferation wasobserved only in the case of coating of the complex formed between anantisense oligonucleotide against HST-1 and a collagen (FIG. 13-5). Onthe other hand, no inhibitory effect was observed in the case of coatingof the complex formed between a sense oligonucleotide against HST-1 anda collagen onto the bottom of the plate (FIG. 13-1). Also, no inhibitoryeffect was observed in the case of coating of the complex formed betweenthe phosphorothioate antisense oligonucleotides having the sequence asscrambling the antisense oligonucleotide sequence (FIG. 13-2) and therandom sequences (FIG. 13-3, 4, 6) and a collagen onto the bottom of theplate. These results show that it is possible to screen for a generesponsible for the cell proliferation without reagents for genetransfer such as cationic liposomes and cationic lipids, which do notusually exist in living bodies, when an antisense oligonucleotidecomplexed with an atelocollagen is coated onto the solid phase, andcells are cultured thereon. Further, these results show that the presentprocess enables to examine sensitively the gene functions even using asmall amount of the cells and the oligonucleotides without noise due todamage to cells from gene transfer reagents.

Example 8 Storage of Adenovirus Vector Under Air Drying UsingAtelocollagen Preparation by Solid Coating

Ten μl of a solution of an adenovirus vector, AdCMVEGFP (K. Aoki, etal., Mol. Ther. (2000) 1 (6): 555-565) at 1×10⁸ pfu/ml in a DMEMsolution was mixed with 5 μl of a solution of an atelocollagen in PBS(−). Fifty μl of the mixture was added to a 24-well culture plate(non-coated) (Corning Inc.), and the plate was air-dried with a dryerset on the cool mood at room temperature for 15 minutes to carry out thesolid coating.

Test of Viruses for Stability During Storage

While the resultant culture plate was left at it was at roomtemperature, a human hepatoma cell line, the HepG2 cells were addedthereto at 2×10⁵ cells/well 1 day, 7 days and 14 days after the leaving,and each 2 days later, the expression level of GFP was observed byfluorescence microscopy. As control, a similar experiment was carriedout using a culture plate coated with only adenovirus, and with only anatelocollagen.

The results are shown in Table 5.

TABLE 5 GFP-positive cells ratio 2 days after the seeding (%)atelo/adeno only adeno only atelo left at r.t.  1 day 64 72 0 left atr.t.  7 days 42 10 or less 0 left at r.t. 14 days r.t. means roomtemperature.

Table 5 shows that the adenovirus vector survived during storage foreven 14 days, suggesting that the solid coating of the present inventionprovides a plate for screening that comprises adenovirus vectors, whichcould be conveniently distributed on the market.

Example 9 Gene Transfer Effects of the Solid Coating in a Dose-DependentManner of Adenovirus

Both equal amounts of a solution of an adenovirus vector AdCMVEGFP (K.Aoki, et al. Mol. Ther. (2000) 1 (6): 555-565) in DMEM at 4×10⁴, 4×10⁵,4×10⁷ pfu/ml, and a 160 μg/ml solution of an atelocollagen were mixedtogether. The mixture could be stored at 4° C. for a long period of timewithout lowered activity. Fifty μl of the mixture was added to a 96-wellculture plate (non-coated; Corning Inc.), and the plate was air-dried tocarry out the solid coating. The plate can be stored at least for 2weeks to 4 weeks without lowered activity. Embryonic stem cells (EScells) were seeded on the resultant culture plate, and three days later,the expression of GFP was checked with fluorescence microscopy,simultaneously with determining the fluorescence intensity. As a result,it was found that the fluorescence intensity of GFP is positivelycorrelated with the dose of adenovirus (FIG. 14). The results shows thatthe present invention should provide a useful tool for high-throughputscreening for ES cells using adenovirus, and a plate for screening,which is coated with complexes formed by adenovirus and collagen.

Example 10 Relationship Between the Composition of Complexes ComprisingPlasmid DNAs and the Average Major Axis

Both equal amounts of an aqueous solution, a 0.1 M phosphate buffer (PB)or a 0.01 M phosphate buffer (PBS) containing sodium chloride, each ofwhich contained a plasmid DNA encoding a fluorescent protein (EGFP)(pCMV-EGFP/pEGFP-N1, 4.7 kbp, Clontech Co.) at 200 μg/ml and was cooledup to 10° C. or less, and an aqueous solution containing anatelocollagen (KOKEN CO., LTD.) at 0.002 to 0.02% (w/w) cooled up to 10°C. or less were mixed together, and the mixtures were left as they wereovernight at 10° C. or less so as to prepare a formulation in a gelform. The gel formulation was penetrated through a filter having a poresize of 70 μm or 10 μm to prepare a gel formulation having an orderedsize.

To the formulation, PicoGreen dsDNA Quantitation Reagent (MolecularProbes) was added to stain pCMV-EGFP, and the major axis of thecomplexes were determined by fluorescent microscopy. Similarly, aplasmid DNA (pCMV-HST-1-IL-2, 6.2 kbp) encoding the secretory signalpeptide of HST-1 and interleukin-2, and an atelocollagen were mixedtogether to prepare a formulation in a gel form, and the major axis ofthe complexes was determined. Further, the gel formulation waselectrophoresed on agarose gel (Tris-acetate buffer, 0.8% agarose gel)to separate plasmid DNAs formed into complexes from those not formedinto complexes. The band of the plasmid DNAs that were not formed intocomplexes was stained with ethidium bromide, and the fluorescenceintensity was determined with Fluor-S-Multi Imager (BIO-RAD). Amount ofthe plasmid DNAs that were not formed into complexes were determined onthe basis of the standard curve obtained from the fluorescence intensityof the band of the plasmid DNA with the known concentration aselectrophoresed, and the ratio of the negative charge of the DNAcontained in the complex to the molecular weight of the collagen. Theresults are shown in Table 6.

TABLE 6 Average major axis of complexes Number of (μm) C.C. N.M perAfter After 70 μm After 10 μm Sample Plasmid DNA Solv. (%) C.M mixingfilter filter 10-1 pCMV-EGFP Water 0.001 2406 9.1 8.9 <3.4 10-2pCMV-EGFP Water 0.005 742 37 26 10-3 pCMV-EGFP Water 0.008 699 6.6 10-4pCMV-EGFP Water 0.01 299 53 44 35 10-5 pCMV-EGFP Water 0.02 356 17 10-6pCMV-EGFP Water 0.05 76 122 105 10-7 pCMV-EGFP Water 0.1 66 303 174 10-8pCMV-EGFP PB 0.001 448 <3.4 <3.4 10-9 pCMV-EGFP PB 0.01 152 7.7 10-10pCMV-EGFP PB 0.1 97 22 14 10-11 pCMV-EGFP PBS 0.01 300 13 9.2 10-12pCMV-EGFP PBS 0.1 97 22 15 10-13 pCMV-HST-1- Water 0.001 4228 6 6.1 <3.4IL-2 10-14 pCMV-HST-1- Water 0.005 1122 20 9.8 IL-2 10-15 pCMV-HST-1-Water 0.008 426 6.1 IL-2 10-16 pCMV-HST-1- Water 0.01 324 20 13 27 IL-210-17 pCMV-HST-1- Water 0.02 360 16 IL-2 10-18 pCMV-HST-1- Water 0.05 95151 201 IL-2 10-19 pCMV-HST-1- Water 0.1 66 256 201 IL-2 10-20pCMV-HST-1- PB 0.001 701 6.1 <3.4 IL-2 10-21 pCMV-HST-1- PB 0.01 262 20IL-2 10-22 pCMV-HST-1- PB 0.1 97 25 9.4 IL-2 10-23 pCMV-HST-1- PBS 0.01315 9.8 IL-2 10-24 pCMV-HST-1- PBS 0.1 97 29 IL-2In the table, “Solv.” means solvent. “C.C. (%)” means collagenconcentration (%). “Number of N.M per C.M” means number of nucleotidemonomer per collagen molecule. “After 70 μm filter” means afterpenetrating through a filter having a pore size of 70 μm. “After 10 μmfilter” means after penetrating through a filter having a pore size of10 μm.

In both cases of pCMV-EGFP and pCMV-HST-1-IL-2, a water without saltswas used as a solvent to obtain complexes wherein the average major axisis 122 μm or more, when collagen concentration was 0.05% or more. Whencollagen concentration was 0.005% to 0.02%, complexes having an averagemajor axis of 6.1 μm to 53 μm were obtained, and when collagenconcentration was 0.001%, complexes having an average major axis of 10μm or less were obtained. The results show that the form or the shape ofa complex can be controlled by adjusting the concentration of a plasmidDNA and a collagen to be mixed, when a water without salts is used as asolvent.

The results are consistent with those as obtained in Example 2 whereinpCAHST-1 was used to prepare complexes. The plasmid DNAs as used thereinwere different each other in size as pCAHST-1 is 7.9 kbp, pCMV-EGFP is4.7 kbp, and pCMV-HST-1-IL-2 is 6.2 kbp, showing that sizes of plasmidDNAs never affect the form or the shape of complexes to be formed.

On the other hand, 0.1 M phosphate buffer and 0.01 M phosphate buffercontaining sodium chloride were used as a solvent to obtain complexeshaving an average major axis of 3.4 μm to 29 μm and no complexes havinga 100 μm or more in size, when collagen concentration was 0.001% to0.1%. It was believed that this was caused by the salts, which inhibitedthe formation of collagen association bodies, showing that whencomplexes are prepared in the presence of an agent that inhibits theformation of collagen association body, then complexes having an averagemajor axis of 100 μm or less can be prepared irrespective of collagenconcentration.

Further, when the gel formulation as obtained by mixing a plasmid DNAand a collagen was penetrated through a filter having a pore size of 70μm or 10 μm, complexes having a smaller average major axis and having anordered size could be prepared.

FIG. 15 shows the relationship between the number of collagen bound toone molecule of plasmid DNA and the average major axis of the complexes.In both cases of pCMV-EGFP and pCMV-HST-1-IL-2 as used in a waterwithout salts, it was found that the average major axis of complexestrends to extend as increasing in the number of collagen bound toplasmid DNA. On the other hand, when 0.1 M phosphate buffer and 0.01 Mphosphate buffer containing sodium chloride were used as a solvent toprepare complexes, such trend as above was not found, and even in thecondition that collagen molecules having an average major axis of 100 μmor more was complexed with a plasmid DNA in case of the use of a wateras a solvent, the average major axis did not exceed 50 μm. It isunderstood that this is caused by extension reaction along the majoraxis of collagen association body, of which formation is developed bythe formation of complexes of a plasmid DNA and a collagen as observedin the case that a water is a solvent as mentioned above. On the otherhand, in case of the presence of salts, it is understood that collagenassociation body formed by attaching a collagen molecule with a plasmidDNA does not extend along the major axis well, and therefore a lot offine association bodies attach to a plasmid DNA to form a structure.Collagens have been known to form fine association bodies at appropriatesalt concentrations. Thus, it can be considered that a partial amount ofcollagens has already formed with a plasmid DNA into fine bodies beforemixing with a plasmid DNA, and the fine collagen association bodies areattached to a plasmid DNA to form complexes. This means that complexeshaving an average major axis of 50 μm or less can be prepared by mixinga plasmid DNA and a collagen that has been formed into fine associationbodies.

For the general analysis of those results regardless of size of plasmidDNAs, the number of nucleotide monomer of a plasmid DNA per one collagenmolecule in complexes was counted. FIG. 16 shows the relationshipbetween the number of nucleotide monomer of plasmid DNAs per onecollagen molecule, and the average major axis of the complexes. It wasfound that the average major axis of complexes trends to extend asdecreasing in the number of nucleotide monomer of plasmid DNAs.

Taken together FIG. 16 and Table 6, the followings are concluded. Whenthe number of nucleotide monomer is 95 or less, the complexes having anaverage major axis of 120 μm or more were obtained. This means that thedecrease in the number of nucleotide monomer per one collagen moleculerepresents the increase in the number of collagen molecules relative tothe plasmid DNA in the complexes, and thus complexes having an averagemajor axis of 120 μm or less can be prepared by keeping the number ofnucleotide monomer of plasmid DNAs per one collagen molecule to be 96 ormore. On the other hand, minimum unit of the complexes is a complexformed by one molecule of collagen and one molecule of plasmid DNA. Onemolecule of collagen and many plasmid DNA hardly form complexes sinceplasmid DNAs repel each other by their negative charge and undergosteric hindrance. Accordingly, the maximum number of nucleotide monomerper one collagen molecule is obtained by forming a complex with each onemolecule of collagen and plasmid DNA, and is defined by the number ofnucleotide monomer comprised in a plasmid DNA.

Example 11

Relationship Between the Composition and the Average Major Axis of theComplex comprising an Oligonucleotide

An equal amount of an aqueous solution, or a 0.1 M phosphate buffer(PB), each of which contained a phosphorothioate antisenseoligonucleotide (⁵′-CTCGTAGGCGTTGTAGTTGT-3′; Molecular weight, about6500; SEQ ID NO: 8) (Espec Inc.) having a sequence complementary to asequence from 4196 by to 4216 by of fibroblast growth factor HST-1(FGF4) gene (described in Proc. Natl. Acad. Sci. USA, 84, 2890-2984(1987)) in a concentration of 2.0 μM to 40.0 μM, and was cooled up to10° C. or less, and an aqueous solution containing an atelocollagen(KOKEN CO., LTD.) at 0.002 to 3.0%(w/w) cooled up to 10° C. or less, 0.1M phosphate buffer were each mixed together at 10° C. or less, and themixtures were left as they were overnight at 10° C. or less so as toprepare formulations in a gel form. Oligonucleotide amounts that werecomplexed with a collagen in the gel formulations were determined asfollows. The gel formulations were centrifuged at 150,000 rotations perhour at 5° C. to precipitate the complexes, and the amounts of theoligonucleotides released in the supernatant were determined by HPLC(column: Puresil C18 5 (Waters), mobile phase: a gradient of 24.5% to35% acetonitrile over 35 minute in 0.1 M ammonium acetate containing 5mM tetrabutyl ammonium and 1mM EDTA, flow rate: 1 ml/min, detectionwavelength: 260 nm). Single-Stranded nucleic acid fluorescence stainingregent YOYO (Molecular Probes) was added to the resultant formulationsto stain the oligonucleotides, which were observed by fluorescentmicroscopy to measure the major axis of the complexes. The results areshown in Table 7.

Average major Number of Number of axis of Sample Solv. O.C. (μM) C.C (%)O.M per C.M N.M per C.M complexes (μm) 11-1 water 1.0 0.05 0.6 12 3211-2 water 5.0 0.05 3 60 13 11-3 water 10.0 0.05 4.4 88 12 11-4 water20.0 0.05 5.2 104 17 11-5 water 10.0 0.01 6.3 126 32 11-6 water 10.0 0.12.9 58 20 11-7 PB 1.0 0.05 0.5 10 25 11-8 PB 5.0 0.05 1.2 24 29 11-9 PB10.0 0.05 1.4 28 35 11-10 PB 20.0 0.05 1.3 26 36 11-11 PB 10.0 0.01 3.366 23 11-12 PB 10.0 0.1 1.3 26 33 11-13 PB 10.0 0.5 0.5 10 46 11-14 PB10.0 1.0 0.3 6 49 11-15 PB 10.0 1.5 0.2 4 54In the table, “Solv.” means solvent. “O.C.(μM) means oligonucleotideconcentration (μM). ” “C.C. (%)” means collagen concentration (%).“Number of O.M per C.M” means number of oligonucleotide molecule per onecollagen molecule. “Number of N.M per C.M” means number of nucleotidemonomer per collagen molecule.

Similarly to Example 10 wherein plasmid DNAs were used, complexes wereobtained by mixing oligonucleotides and collagens. In both cases of useof a water without salts and 0.1 M phosphate buffer as a solvent, therewere no significant change in major axis of the complexes between thedifferences in the concentrations of collagen and oligonucleotide, whichwas different from the case of plasmid DNAs. This is believed to becaused by the fact that oligonucleotides are smaller than plasmid DNAsin molecular size, and thus the formation of collagen association bodiesis inhibited.

FIG. 17 shows the relationship between the molecular number ratio ofoligonucleotide to collagen at the time of the mixture and the number ofoligonucleotide bound to one molecule of collagen in the complex. It wasfound that the number of oligonucleotides bound to one collagen moleculein the formed complexes trends to increase as increasing in the numberof oligonucleotides relative to the number of collagen molecule at thetime of mixing in both cases that a water and the phosphate buffer wereused as a solvent. The maximum number of oligonucleotide molecule boundto one collagen molecule varies depending on kinds of solvent, and it is6 molecules in case of water, whereas it is 3 molecules in case of thephosphate buffer.

Since molecular weight of a collagen (300,000) is even larger than thatof an oligonucleotide (about 6,500), and the form or the shape ofcomplexes depends solely on the number of collagen molecule forming intocomplexes, the numbers of collagen molecule in complexes that have thesame major axis are approximately the same. Accordingly, even for thecomplexes having the same major axis, the number of oligonucleotidemolecule comprised in complexes varies depending on the number ofoligonucleotide bound to one collagen molecule. Because individualcomplex contacts with a cell to allow oligonucleotides to be transferredinto the cell, oligonucleotide transfer is more efficient when a complexthat contains a high content of oligonucleotide molecules is used.Accordingly, a complex wherein the number of oligonucleotide moleculeper one collagen molecule is increased is desired, and generallyspeaking, a complex wherein the number of nucleotide monomer bound toone collagen molecule is increased is preferred.

Example 12

Cell Culture Instrument Coated with a Complex Comprising anOligonucleotide

Fifty μl of the gel formulation as prepared in Example 11 (11-3, thecomposition of the formulation of Example 7) was added dropwise to thebottom of a 96-well microplate, and dried by directly spraying a coolair (room temperature, 2 hours) or by placing in a desicator with silicagel (room temperature, 2 days) so as to prepare a cell cultureinstrument, of which the cell culture surface is coated with a complex.One hundred μl of 0.01 M phosphate buffer (PBS) that had been adjustedto be isotonic with living bodies by supplementing sodium chloride wasadded to the wells of the instrument, and the pipetting was slightlyconducted immediately for sampling. Further, 100 μl of 0.01 M phosphatebuffer (PBS) that had been adjusted to be isotonic with living bodies bysupplementing sodium chloride was added to the wells of the instrument,and the instrument was left overnight at 37° C., followed by slightlypipetting for sampling. To 10 μl of the resultant sample, 2 μl ofSingle-Stranded nucleic acid fluorescence staining regent YOYO(Molecular Probes) was added to stain the oligonucleotides, and thecomplexes that released form the surface of the instrument were observedby fluorescent microscopy. The fluorescence micrograph is shown in FIG.18. The complexes having a major axis of 50 μl or less were observed inall of the samples. This means that exposure on PBS allows to releasecomplexes from the surface of a cell culture instrument. The form or theshape of the released complexes was not changed between before and afterthe coating onto the surface of the instrument irrespective of dryingmethod, and condition of PBS exposure. This shows that the complexes areretained on the surface of a cell culture instrument with keeping theirform or shape irrespective of drying method, and that the form or theshape of the complexes maintains in a temperature condition of 37° C.that is usually used in cell culture.

Example 13

Cell Culture Instrument Coated with a Complex Comprising a Plasmid DNA

Fifty μl of the gel formulation as prepared in Example 10 (10-3, havingthe composition that was found efficient in transfer efficiency inExample 6) was added dropwise to the bottom of a 96-well microplate, anddried by directly spraying a cool air (room temperature, 2 hours) or byplacing in a desicator with silica gel (room temperature, 2 days) so asto prepare a cell culture instrument, of which the cell culture surfaceis coated with a complex. One hundred μl of 0.01 M phosphate buffer(PBS) that had been adjusted to be isotonic with living bodies bysupplementing sodium chloride was added to the wells of the instrument,and the pipetting was slightly conducted immediately for sampling.Further, 100 μl of 0.01 M phosphate buffer (PBS) that had been adjustedto be isotonic with living bodies by supplementing sodium chloride wasadded to the wells of the instrument, and the instrument was leftovernight at 37° C., followed by slightly pipetting for sampling. To 10μl of the resultant sample, 2 μl of Single-Stranded nucleic acidfluorescence staining regent YOYO (Molecular Probes) was added to stainthe oligonucleotides, and the complexes that released form the surfaceof the instrument were observed by fluorescent microscopy. Thefluorescence micrograph is shown in FIG. 19. The complexes having amajor axis of 100 μl or less were observed in all of the samples. Thismeans that exposure on PBS allows to release complexes from the surfaceof a cell culture instrument. The form or the shape of the releasedcomplexes was not changed between before and after the coating onto thesurface of the instrument irrespective of drying method, and conditionof PBS exposure. This shows that the complexes are retained on thesurface of a cell culture instrument with keeping their form or shapeirrespective of drying method, and that the form or the shape of thecomplexes maintains in a temperature condition of 37° C. that is usuallyused in cell culture.

Example 14

Gene Transfer with a Cell Culture Instrument Coated with a Complex

Three hundreds μl of the gel formulations as prepared in Example 10,10-1, 10-4, and 10-7, comprising pCMV-EGFP were added to a 6-well cellculture microplate, and dried by spraying a cool air so as to prepare acell culture instrument onto which a complex comprising pCMV-EGFP wascoated. As control, 300 μl of an aqueous solution comprising pCMV-EGFPat 100 μm/ml was added to a plate, and dried similarly. Into each well,7.5×10⁴ cells of 239 cells were seeded, and a DMEM medium containing 10%FBS was added thereto, followed by culturing at 37° C. From the start ofthe culture, the medium was replaced with a fresh medium every 4 or 5days. eleven days after the seeding, the cells were observed byfluorescent microscopy, and the number of the cells expressing GFP wascounted to estimate transfer efficiency.

Similarly, 300 μl or 500 μl of the gel formulations as prepared inExample 10, 10-13 to 22, and 24, comprising pCMV-HST-1-IL-2 were addedto a 6-well cell culture microplate, and dried by spraying a cool air soas to prepare a cell culture instrument onto which a complex comprisingpCMV-HST-1-IL-2 was coated. As control, 300 μl or 500 μl of an aqueoussolution comprising pCMV-HST-1-IL-2 at 100 m/ml, PB solution or PBSsolution were added to a plate, and dried similarly.

1) Transfer of Plasmid DNA into 239 Cells

Into each well wherein 300 μl of the gel formulation was coated, 7.5×10⁴cells of 239 cells were seeded, and a DMEM medium containing 10% FBS wasadded thereto, followed by culturing at 37° C. for 10-13 to −19. Eightdays after the cell seeding, the medium was replaced, and IL-2concentration in the medium sampled 11 days later was determined byELISA (Quamtikine human IL-2 (R&D Systems)). For 10-20 to −22, a DMEMmedium was added to the plate, and cultured overnight at 37° C., afterwhich on the following day the medium was replaced with a DMEM mediumcontaining 10% FBS, and then cultured at 37° C. Eight days and 11 daysafter the cell seeding, the medium was replaced, and IL-2 concentrationin the medium sampled 15 days later was determined by ELISA. Further,for 10-20, 21, and 24, a DMEM medium containing 10% FBS was added to theplate, and the plate was cultured at 37° C. IL-2 concentration in themedium sampled 8 days after the cell seeding was determined by ELISA.

2) Transfer of Plasmid DNA into NIH3T3 Cells

Into the well wherein 500 μl of 10-14 to -17 was coated, 5×10⁴ cells ofNIH3T3cells were seeded, and a DMEM medium containing 10% FBS was addedthereto, followed by culturing at 37° C. Eight days after the cellseeding, the medium was sampled, and IL-2 concentration therein wasdetermined by ELISA.

The results as obtained in above 1) and 29 are summarized in Tables 8and 9. When complexed with a collagen and coated onto a cell cultureinstrument, both pCMV-EGFP and pCMV-HST-1-IL-2 could be efficientlytransferred into 293 cells and NIH3T3 cells. The result shows that theeffect of the complexes of the present invention on facilitation of genetransfer is not affected by the kinds of plasmid DNA, species of thecells, and the presence or the absence of serum in the culture. Further,in case of plasmid DNAs, it has been found that coating of the complexhaving 1112 or less of the nucleotide monomer per one collagen molecule,and having an average major axis of 151 μm or less, provided superiortransfer efficiency and gene expression.

TABLE 8 Number of nucleotide monomer per collagen Average major axisSample molecule of complexes (μm) transfer efficiency Control 0.002810-1 2406 9.1 0.0760 10-4 299 53 0.0729 10-7 66 303 0.0374

TABLE 9 Number of Presence nucleotide Average IL-2 or monomer major axisof concentration in Cell Absence per collagen complexes media Samplespecies Solvent of FBS molecule (μm) (pg/ml) Control 293 water Presence5.3 10-13 293 water Presence 4228 6 7.3 10-14 293 water Presence 1122 2014.8 10-15 293 water Presence 426 6.1 19.3 10-16 293 water Presence 32420 6.3 10-17 293 water Presence 360 16 18.2 10-18 293 water Presence 95151 8.4 10-19 293 water Presence 66 256 5.3 Control 293 PB Absence 23.5510-20 293 PB Absence 701 6.1 72.93 10-21 293 PB Absence 262 20 264.6110-22 293 PB Absence 97 25 86.22 Control 293 PB Presence 3.35 10-20 293PB Presence 701 6.1 21.01 10-21 293 PB Presence 262 20 86.68 Control 293PBS Presence 1.07 10-23 293 PBS Presence 315 9.8 15.26 Control NIH3T3water Presence 0 10-14 NIH3T3 water Presence 1122 20 0.79 10-15 NIH3T3water Presence 426 6.1 6.06 10-16 NIH3T3 water Presence 324 20 4.0510-17 NIH3T3 water Presence 360 16 2.78

Example 15

Gene Transfer into Cells by Addition of Complexes

Into a 6-well plate, 5×10⁴ cells of 239 cells were seeded, and culturedin the presence of a DMEM medium containing 10% FBS at 37° C. Three daysafter the seeding, 300 μl of the gel formulations as prepared in Example10, 10-1, 10-4, 10-7, 10-8, 10-9, and 10-10, comprising pCMV-EGFP, and awater or a PB solution comprising pCMV-EGFP at 100 μg/ml as control wereeach added thereto, and the cells were cultured in the presence of aDMEM medium containing 10% FBS at 37° C. On the following day, the mediawere replaced with a fresh medium, and 13 days later for 10-1, 10-4, and10-7, or 5 days later for 10-8, 10-9, and 10-10, the cells were observedby fluorescent microscopy, followed by counting the number of the cellsexpressing GFP.

Similarly, 7.5×10⁴ cells of 239 cells were seeded into a 6-well plate,and cultured in the presence of a DMEM medium containing 10% FBS at 37°C. Two days after the seeding, 300 μl of the gel formulations asprepared in Example 10, 10-20 and 10-21, comprising pCMV-HST-1-IL-2, anda PB solution comprising pCMV-HST-1-IL-2 at 100 μg/ml as control wereeach added thereto, and the cells were cultured in the presence of aDMEM medium containing 10% FBS at 37° C. On the following day, the mediawere replaced with a fresh medium, and 5 days later, the media weresampled, and IL-2 concentration therein were determined by ELISA(Quamtikine human IL-2 (R&D Systems)).

The results are summarized in Tables 10, 11 and 12. When complexed witha collagen and added to cells, both pCMV-EGFP and pCMV-HST-1-IL-2 couldbe efficiently transferred into the cells. It has been found that incase of the complexes having an average major axis of 53 μm or less asshown in the present Example, the addition of the complex having 2406 orless, and further 701 or less of the nucleotide monomer per one collagenmolecule, provided superior transfer efficiency and gene expression.

TABLE 10 Number of nucleotide Average major monomer per collagen axis ofcomplexes transfer Sample Solvent molecule (μm) efficiency Control water0.0012 10-1 water 2406 9.1 0.0231 10-4 water 299 53 0.0247 10-7 water 66303 0.0041

TABLE 11 Average Number of nucleotide major axis Number monomer percollagen of complexes of EGFP- Sample Solvent molecule (μm) expressingcells Control PB 100 10-8 PB 448 <3.4 128 10-9 PB 152 7.7 1200 10-10 PB97 22 12000

TABLE 12 Average IL-2 Number of nucleotide major axis concentrationmonomer per collagen of complexes in Sample Solvent molecule (μm) media(pg/ml) Control PB 0 10-20 PB 701 6.1 8.98 10-21 PB 262 20 76.81 10-22PB 97 25 120.74

INDUSTRIAL APPLICABILITY

According to the complexes of the present invention, it is possible topreserve and stabilize desired nucleic acids by complexing withcollagens or collagen derivatives, 2) to transfer efficiently desirednucleic acids into cells when administered to living bodies, and 3) toexpress and inhibit desired nucleic acids without reagents for genetransfer. The complexes of the present invention can be applied tovarious aspects since the complexes of the invention have advantages 1)that DNAs, antisense oligonucleotides and virus vectors can be preservedand stabilized, 2) that the complexes can be stored during a long periodof time on plates dried where the complexes are coated, 3) that genescan be expressed without reagents for gene transfer when cells areseeded onto the plates, 4) that kinds of subjects of nucleic acids andmethods for immobilization are not limited to specific ones, and 5) thatthe duration time of the expression in case of plasmid DNAs isexcellently extended.

According to the present invention, it is possible to readily examinethe functions of genes or proteins, of which the expressions areinhibited by antisense oligonucleotides by determining the proliferationor the morphology of the cells, or the expression levels of cytokines orreceptors. Not only antisense oligonucleotides, but also other materialscan be complexed with collagens to form complexes, which in turn can becoated onto plates; in case of ribozymes, it is possible to carry out ascreening similar to that of antisense oligonucleotides; and further incase of plasmid DNAs or adenoviruses, it is possible to examine thefunctions of genes or proteins by determining the changes of cells bythe expression of certain genes. Collagens or collagen derivativescomprised in the complexes of the present invention have been known toaffect no cells, and the screening using the complexes of the presentinvention makes it possible to carry out the examinations of genefunctions that seldom avoid the unspecific affections to cells, whichwould be induced by conventional reagents for gene transfer.

1. A method of transferring an oligonucleotide to a target organ or atarget tissue comprising administering an electrostatic complexconsisting essentially of an oligonucleotide and a water-solubleatelocollagen by injection.
 2. The method according to claim 1, whereinthe oligonucleotide is 5-30 nucleotides in length.
 3. The methodaccording to claim 1, wherein the ratio of the number of atelocollagenmolecules to the number of nucleotide monomers of oligonucleotide is1:20 to 1:120.
 4. The method according to claim 1, wherein the size ofthe electrostatic complex is 300 nm to 30 um.
 5. The method according toclaim 3, wherein the oligonucleotide is a DNA derivative or an RNAderivative.
 6. The method according to claim 3, wherein theoligonucleotide is a DNA derivative or an RNA comprising aphosphorothioate bond.
 7. The method according to claim 3, wherein theoligonucleotide is a DNA derivative or an RNA comprising aninternucleotide comprising a phosphate, sugar or base moiety chemicallymodified to avoid enzymatic degradations.
 8. The method according toclaim 1, wherein the complex exhibits a major axis of about 300 nm inlength.
 9. The method according to claim 1, wherein the complex exhibitsa major axis from 12 to 54 microns in length.
 10. The method accordingto claim 1, wherein the ratio of the number of oligonucleotide moleculesto the number of atelocollagen molecule is from 0.2 to 6.3.
 11. Themethod according to claim 1, wherein the ratio of the nucleotidemonomers per the number of atelocollagen molecules is from 4 to 126.